Silibinin suppresses growth and induces apoptotic death of human colorectal carcinoma LoVo cells in culture and tumor xenograft

Abstract

Colorectal cancer is one of the leading causes of cancer-related morbidity and mortality. The use of nontoxic phytochemicals in the prevention and intervention of colorectal cancer has been suggested as an alternative to chemotherapy. Here we assessed the anticancer efficacy of silibinin against advanced colorectal cancer LoVo cells both in vitro and in vivo. Our results showed that silibinin treatment strongly inhibits the growth of LoVo cells (P < 0.05-0.001) and induces apoptotic death (P < 0.01-0.001), which was associated with increased levels of cleaved caspases (3 and 9) and cleaved poly(ADP-ribose) polymerase. Additionally, silibinin caused a strong cell cycle arrest at G(1) phase and a slight but significant G(2)-M-phase arrest at highest concentration (P < 0.01-0.001). Molecular analyses for cell cycle regulators showed that silibinin decreases the level of cyclins (D1, D3, A and B1) and cyclin-dependent kinases (1, 2, 4, and 6) and increases the level of cyclin-dependent kinase inhibitors (p21 and p27). Consistent with these results, silibinin treatment also decreased the phosphorylation of retinoblastoma protein at Ser(780), Ser(795), and Ser(807)/Ser(811) sites without significantly affecting its total level. In animal studies, oral administration of silibinin for 6 weeks (at 100 and 200 mg/kg/d for 5 days/wk) significantly inhibited the growth of LoVo xenograft (P < 0.001) in athymic nude mice without any apparent toxicity. Analyses of xenograft tissue showed that silibinin treatment inhibits proliferation and increases apoptosis along with a strong increase in p27 levels but a decrease in retinoblastoma phosphorylation. Together, these results suggest the potential use of silibinin against advanced human colorectal cancer.

Fig. 1. Silibinin inhibits the growth and decreases the viability of human CRC LoVo cells

LoVo cells were plated overnight and subsequently treated with silibinin at concentrations ranging from 0–200 μM in DMSO for 24–72 h. At the end of each treatment duration, cells were collected and processed for the determination of live cell number (A) and dead cells number (B) as mentioned in the Materials and methods. The data shown are mean ± standard deviation of three samples for each treatment. Experiment was repeated three times. *, P < 0.05; #, P < 0.01; $, P < 0.001

Fig. 2. Silibinin induces apoptotic death in human CRC LoVo cells

LoVo cells were plated overnight and subsequently treated with 0, 100 and 200 μM silibinin in DMSO for 24 and 48 h. At the end of each treatment duration, both adherent and non-adherent cells were collected and processed. (A) Collected cells were stained with Annexin V-PI and analyzed with flow cytometer and presented as percent apoptotic death. The data shown are mean ± standard deviation of three samples for each treatment. In each case experiment was repeated three times. #, P < 0.01; $, P < 0.001. (B) Total cell lysates were prepared at the end of the treatment as described in Materials and methods, and separated on SDS-PAGE. Separated proteins were then immunoblotted with antibodies for cleaved caspase-9, -3 and cleaved PARP. Membranes were also stripped in each case and reprobed with anti-β-actin antibody to confirm equal protein loading.

Fig. 3. Silibinin causes cell cycle arrest and modulates the expression of various cell cycle regulatory molecules in human CRC LoVo cells

LoVo cells were plated overnight and subsequently treated with silibinin at concentrations ranging from 0–200 μM in DMSO for 24 and 48 h. At the end of treatment durations, cells were collected, and were either (A and B) stained with saponin-PI and analyzed for cell cycle distribution by flow cytometry, or (C and D) total cell lysates were prepared and analyzed for various cell cycle regulatory molecules (CDK1, CDK2, CDK4, CDK6, cyclin D1, cyclin D3, cyclin A, cyclin B1, p21, p27, phosphorylation of Rb at Ser 795, Ser 780, Ser 807/811 and total Rb) by western immunoblotting. Membranes were also stripped in each case and reprobed with anti-β-actin antibody to confirm equal protein loading. #, P < 0.01; $, P < 0.001

Fig. 4. Silibinin inhibits human CRC LoVo xenograft growth in athymic nude mice

Each mouse was s.c. injected with 5 × 10⁶ LoVo cells mixed with matrigel (1:1) on the right flank. After 24 h, mice were gavaged with CMC (control group) or 100 and 200 mg/kg body weight/day doses of silibinin for 5 days/week for 6 weeks: (A) Tumor volume (mm³) per mouse as a function of time. (B) Tumor weight (grams) per mouse at the end of study. (C) Average body weight (grams) per mouse. (D) Average diet consumption (grams) per mouse per day. In panels A and B, data shown are mean ± SE from 8 mice in each group. Abbreviations: SB-100, 100 mg/kg body weight silibinin; SB-200, 200 mg/kg body weight silibinin.

Fig. 5. In vivo antiproliferative and pro-apoptotic effect of silibinin in human CRC LoVo xenograft in athymic nude mice

(A and B) At the end of the experiment mice were sacrificed and tumor tissues were analyzed for immunohistochemical staining of proliferating cell nuclear antigen (PCNA) and photomicrographs were taken as detailed in the Materials and methods. Proliferation index was calculated as number of PCNA positive cells × 100/total number of cells counted under 400X magnification in 5 randomly selected areas in each tumor sample. (C and D) Apoptotic cell population in tissues from various groups was analyzed by TUNEL assay as detailed in the Materials and methods. Apoptotic index was calculated as number of positive cells × 100/total number of cells counted under 400× magnification in five randomly selected areas in each tumor sample. Data shown represents mean ± SE from 8 mice in each group. Abbreviations: SB-100, 100 mg/kg body weight silibinin; SB-200, 200 mg/kg body weight silibinin.

Fig. 6. Silibinin modulates the expression of cell cycle regulatory molecules in human CRC LoVo xenograft in athymic nude mice

Three randomly selected samples from each group were analyzed for cell cycle regulatory molecules. Total cell lysates were prepared and western blot analysis was performed for p27, phospho-Rb (Ser 795 and Ser 807/811) and total Rb as detailed in the Materials and methods. Membranes were also stripped in each case and reprobed with anti-β-actin antibody to confirm equal protein loading. Abbreviations: Con, Control; SB- 100, 100 mg/kg body weight silibinin; SB-200, 200 mg/kg body weight silibinin.